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Human Skeletal Muscle Proteins and Metabolic Pathways are Susceptible to Dicarbonyl Stress
Author(s) -
Mey Jacob T,
Haus Jacob M
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.710.6
Subject(s) - methylglyoxal , ampk , skeletal muscle , medicine , arginine , chemistry , endocrinology , glycation , glucose uptake , amp activated protein kinase , carbohydrate metabolism , metabolism , glycolysis , biochemistry , protein kinase a , diabetes mellitus , biology , enzyme , insulin , amino acid
Individuals with Type 2 Diabetes Mellitus (T2DM) present with hyperglycemia concomitant with increased generation of methylglyoxal (MG), a highly reactive dicarbonyl and glycolytic intermediate. MG modifies proteins at arginine residues creating the advanced glycation endproduct methylglyoxal‐derived hydroimidazolone (MG‐H1). Accumulation of MG‐modified proteins has been referred to as dicarbonyl stress and has been implicated in the etiology of T2DM and development of future complications. The mechanisms and tissue specificity underlying MG‐directed protein modifications are largely unknown, but MG has been hypothesized to target the regulatory gamma subunit of AMPK (AMPKγ), due to exposed arginine residues within the AMPKγ catalytic site. PURPOSE Therefore, the purpose of this study was to investigate the effects of hyperglycemia or exogenous MG treatment on the formation of total MG‐modified proteins and specific AMPKγ protein modifications in primary human skeletal muscle cells (HSKMCs). Skeletal muscle tissue was selected given the paucity of evidence and the importance to whole body glucose metabolism. METHODS HSKMCs were incubated in euglycemic (5.5mM glucose) or hyperglycemic (30mM glucose) media and total MG‐directed protein modifications were assessed at 24 hrs and 5 days in cell lysates via immunoblotting of MG‐H1 residues. Specific MG‐modifications of AMPKγ were assessed by incubating HSKMCs in MG‐supplemented media (0, 5, 50, 100, or 500μM MG) for 4 hours after which AMPKγ was immunoprecipitated and immunoblotted for MG‐H1 residues. We also examined the effects of MG treatment on AMPK activity, with and without AICAR stimulation (2mM), as assessed by the expression of phosphorylated acetyl coa carboxylase β (pACCβ), a commonly used surrogate marker for AMPK activity in skeletal muscle. RESULTS Hyperglycemia showed no effect on total MG‐modified proteins at 24 hrs (p>0.05), but increased MG‐modified proteins by 50±18% (p<0.035) at 5 days. Experimental MG treatment did not increase MG‐modifications of AMPKγ at any MG concentration. However, ACCβ phosphorylation increased in stepwise fashion with increasing MG concentrations, becoming significant at 10μM (141±4%, p=0.0066), 50 μM (202±9%, p=0.0007) and peaking at 100μM (342±27%, p=0.0010). Interestingly, pre‐incubating HSKMCs with low dose MG (10μM) was additive to AICAR stimulated AMPK activity as the combination increased pACCβ expression by 24±4% compared to AICAR alone (p=0.011). However, pre‐incubating cells with MG at high doses showed no additional effect or abolished pACC expression (500μM, no different from Control). CONCLUSION These data suggest that hyperglycemia increases dicarbonyl stress in HSKMCs and that exogenous MG treatment augments AMPK activity in the absence of AMPKγ specific MG‐modifications. The biphasic response in AMPK activity in response to exogenous MG requires further investigation.